2000 Oxford University Press

Nucleic Acids Research, 2000, Vol. 28, No. 12

e67

A simple and efficient method for PCR amplifiable DNA

extraction from ancient bones
Tibor Kalmr*, Csand Z. Bachrati, Antnia Marcsik1 and Istvn Rask
Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, POB 521, H-6701, Szeged,
Hungary and 1Department of Anthropology, University of Szeged, Egyetem u. 2, H-6725, Szeged, Hungary
Received March 7, 2000; Revised and Accepted April 27, 2000

ABSTRACT
A simple and effective modified ethanol precipitationbased protocol is described for the preparation of
DNA from ancient human bones. This method is fast
and requires neither hazardous chemicals nor
special devices. After the powdering and incubating
of the bone samples Dextran Blue was added as a
carrier for removing the PCR inhibitors with selective
ethanol precipitation. This method could eliminate
the time-consuming separate decalcification step,
dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR
amplification. The efficiency of this procedure was
demonstrated on ten 5001200-year-old human
bones from four different Hungarian burial sites. A
mitochondrial specific primer pair was used to obtain
sequence information from the purified ancient DNA.
The PCR amplification, after our DNA extraction
protocol, was successful from each of the 10 bone
samples investigated. The results demonstrate that
extraction of DNA from ancient bone samples with
this new approach increases the success rate of PCR
amplification.
INTRODUCTION
Extraction and successful PCR amplification of DNA from
human remains in historical and forensic cases has great
importance, but is particularly difficult because the methods
employed at present are not always satisfactory. Previous
studies have shown that DNA can persist in ancient remains
and the best subjects for such investigations are bone and tooth
samples since they are much more abundant than soft tissue
remains and generally better preserved (1,2).
The factors that commonly prevent PCR amplification of
DNA from ancient remains may vary between burial sites.
They may originate either from the environment of the remains
in the form of humic acid, fulvic acid, hidroxi-apatite, tannin
and contaminating DNA, or from degradation in the biological
sample (39). Ancient DNA is heavily modified and these
modifications, which are mainly attributed to oxidative processes,
are responsible for the low recovery rate of undamaged DNA
from archaeological specimens (4,10). In the case of bone,

DDBJ/EMBL/GenBank accession nos AF228540AF228549

collagen type I (11) and Maillard products (2,4,12) are the

main inhibitory factors of successful PCR amplifications.
The first steps of DNA extraction in the majority of the previously published methods were the powdering of bone material
and incubation in various extraction buffers (2,4,1316). Classically, in the next step the DNA was extracted with phenolchloroform (2,4,7,15,1720) or the extract was dialysed against
EDTA and TrisHCl buffered solution (2,7). After extraction,
the aqueous phase was concentrated by means of ethanol or
isopropanol precipitation (2,16,17,21), or microconcentrators
(4,7,20,22). Alternatively, after the incubation step, DNA could
be separated with glass-milk or silica suspension and could be
eluted from silica pellet (17,21,2325). The Chelex-based method
involved boiling the bone powder in Chelex suspension, followed
by PCR amplification of the supernatant (18,26).
Experiments that compared the phenol-chloroform and
Chelex techniques concluded that although the Chelex method
was simple and fast, inhibitory substances had not been
eliminated in most of the cases (27). Another study showed
that sodium-acetate-isopropanol extraction was possibly better
than the phenol-chloroform method and resulted in about three
times the quantity of extracted DNA than the glass-milk
method (21). According to the available data the isopropanolbased method seems to be the most efficient technique for
extracting DNA from ancient remains.
A simple and useful carrier-mediated ethanol precipitationbased method is described here for the extraction of PCR
amplifiable DNA from ancient bones. According to our judgement this protocol is superior to the previous techniques; it has
the advantages of the isopropanol-based method and, in
addition, the success rate of extraction is higher and results in
DNA free from inhibitory impurities. It does not involve
hazardous organic solvents, special devices or numerous
enzymes, which increase the possibility of contamination and
DNA degradation (4,22,28,29). The efficiency of this protocol
is demonstrated on 10 human bone samples originating from
the 7th to 15th centuries.
MATERIALS AND METHODS
Samples
The bones were provided by the Department of Anthropology
(University of Szeged, Hungary) and derived from four
different well-documented Hungarian excavations from 7th15th
century cemeteries (3033). The gender of the remains was

*To whom correspondence should be addressed. Tel: +36 62 432 080; Fax: +36 62 433 503; Email: klampar@nucleus.szbk.u-szeged.hu

e67

Nucleic Acids Research, 2000, Vol. 28, No. 12

Table 1. The origin, age, sex and type of the investigated 10 bone samples

denotes where a feature is unknown.

Table 2. Primers used in this study

The letters H and L refer to the heavy and light strands of the mtDNA and the numbers refer to the position
of the 5 base of the primer in the complete human mitochondrial DNA sequence (35). 239 and 406 bp fragments
from the hypervariable region I were amplified with primer pairs H16420L16182 and H16401L15996,
respectively.

determined by anthropometric evaluations. The other characteristics of the bone samples are listed in Table 1.
Contamination precaution
In order to prevent possible contamination all stages of the
work were carried out under sterile conditions, using latex
gloves, mouth masks and Plexiglas facemasks. All appliances,
containers and the work areas (laminar airflow surface, PCR
box) were cleaned and irradiated with 1.0 J/cm2 UV-C light for
at least 60 min. The extraction buffer (without proteinase K),
Dextran Blue solution, NH4-acetate and deionised, distilled
water were irradiated for 30 min. All steps (bone cutting,
surface removing, powdering, extraction and amplification)
were carried out in separate places. Throughout all manipulations Aer ultra micro sterile tips (ELKay, Costelloe, Ireland)
were used for pipetting.
DNA extraction
Surface material was removed from the bones by washing with
diluted bleach and distilled water. A 2 5 cm portion was cut
from each bone diaphysis, and the surfaces of these portions
were removed (at least 23 mm deep) with a sand disk in order
to get rid of modern DNA contamination. The cleaned bone
fragments were treated with UV light at 1.0 J/cm2 for 30 min,
and mechanically ground into a fine meal in a sterile agate
mortar. Physically powdered bone (750 mg) was suspended in
ii

1.6 ml extraction buffer (0.1 M EDTA, 0.5% N-laurylsarcosine-Na salt, 100 mg/ml proteinase K), vortexed and incubated
overnight at 37C with continuous vertical rotation. After
phase separation by centrifugation at room temperature at
12 000 r.p.m. for 10 min, 250 l supernatant was transferred to
a 1.5 ml Eppendorf tube and 3.5 l 1 g/l Dextran Blue
(Sigma, Budapest, Hungary), 250 l 4 M NH4-acetate and
500 l 96% EtOH were added and mixed by vortexing.
Dextran Blue has large size (greater than 2 million molecular
mass), effectively coprecipitates low concentrations of DNA
and colours the pellet. PCR is inhibited in a dose-dependent
manner at concentrations of Dextran Blue only >125 g/ml. It
remains in the well during the gel run and thus does not
interfere with sequence recordings (34).
The DNA was precipitated at 70C for 7 min and centrifuged at 14 000 r.p.m. at 4C for 15 min. The pellet was redissolved in 2030 l deionised, distilled water. The remaining
extract was stored at 20C.
Amplification
A typical amplification reaction contained 27 l of bone
extract, 1 U Taq DNA polymerase (Zenon Biotechnology Ltd,
Szeged, Hungary), 160 g/ml BSA (Boehringer Mannheim,
Mannheim, Germany), 200 M each of dNTP (Boehringer
Mannheim), 20 pmol each of mtDNA-specific primers
(Table 2), 1 PCR Buffer (Perkin Elmer Cetus, Budapest,

Nucleic Acids Research, 2000, Vol. 28, No. 12

e67

Hungary) in 25 l total volume. Denaturation was at 93C for

5 min, followed by 35 cycles of denaturation at 93C for 1 min,
annealing at 58C for 1 min and extension at 72C for 1 min.
The last cycle was followed by an extra extension step at 72C
for 5 min. Amplification reactions were prepared in a PCR box
using dedicated pipettes that had never been in contact with
amplified DNA.
The efficiency and reliability of PCR reactions was monitored
by two parallel control reactions: an extraction control to check
the purity of the mock DNA extraction with no bone added and
an amplification control to check the purity of the PCR
reagents with no DNA added.
In order to check the fragment size and quality of amplification
one-fifth of the PCR product was run on a 5% native polyacrylamide gel.
Sequencing
After successful and contamination-free amplification, threefifths of the volume of the PCR products were run on a 1.5%
agarose gel. The specific bands were cut out from the gel,
recovered and 90120 ng redissolved PCR products were
directly sequenced with ABI Prism 310 sequencer (Perkin
Elmer). The primers for sequencing were the same as those
used for amplification.
RESULTS AND DISCUSSION
Hnni et al. published an isopropanol-based precipitation
method (2) that could eliminate the time-consuming dialysis of
the bone DNA extract and the concentration step by centrifugation-driven microconcentrator. It was found that isopropanol precipitation after the phenol-chloroform extraction step
strongly reduced the blurring blue fluorescence present on
agarose gels. It was also demonstrated that the intensity of blue
fluorescence (which was derived from Maillard products of
reducing sugars) showed strong correlation with PCR inhibitory activity. The authors however noted that they could find
various levels of remaining blue fluorescence and PCR
inhibitory activity in samples extracted by their method.
Similarly in our experiments, using the previously published
method shown in Figure 1A, Maillard products were present in
the DNA extract from ancient bone. Besides the presence of
Maillard products, the extract produced inhibitory activity in
PCR reactions of control DNA as seen in Figure 1B. The inhibition was decreased by increased dilution of the extract. These
impurities could prevent the successful amplification of mitochondrial DNA fragments from ancient bone samples. As
depicted in Figure 1C, where the electrophoretic profile of the
DNA isolated by our method is shown, there is no detectable
blue fluorescence on the agarose gel and the length of the
majority of intact template molecules is <400 bp. The extract
produced by this new method has no inhibitory activity in PCR
reactions as seen in Figure 1D. The general lack of inhibition
in our extracts could be due to the removal of the bone surfaces
and the application of Dextran Blue which precipitates only the
DNA.
As depicted in Figure 2A a 239 bp mtDNA fragment from
the hypervariable region I was successfully amplified from
ancient bone DNA extracted by our new method but failed
using the isopropanol extraction protocol. The amplification
with 35 cycles of this fragment was successful from each of the

Figure 1. Comparison of the presence of Taq inhibitor(s) in the DNA extract

purified by the previously published isopropanol-based and the new carriermediated methods and determination of the degree of degradation of extracted
ancient bone DNA. (A) The inhibitors destroyed the structure of the 1%
agarose gel. Lane MW, Lambda/HindIII molecular weight marker; lane B3,
ancient bone (no. 3) extract. Under the UV-light the bulb could be seen as a
blue cloud formed by the Maillard-product. (B) Amplification-inhibitory
effect of the DNA extract from ancient bone no. 3 produced by the isopropanolbased method. By increasing the amount of bone extract a reduction of the
406 bp fragment from a blood sample was observed (05 l bone extract in
50 l total reaction volume, in lanes 05, respectively). Lane MW, 100 bp
molecular weight marker (MBI Fermentas, Vilnius, Lithuania). (C) Fragmented
ancient bone DNA extracted by carrier-mediated ethanol-based precipitation
method, compared to DNA from whole blood and hair extracted by phenolchloroform extraction method after digestion with proteinase K. Lane MW,
100 bp molecular weight marker (MBI Fermentas); lanes B3 and B7, DNA
extracted from bone no. 3 and 7, respectively. (D) Amplification-inhibitory
effect of the DNA extract from ancient bone no. 3 by our new protocol. By
increasing the amount of bone extract no effect of the amount of the 406 bp
fragment from a blood sample was observed (05 l bone extract in 50 l total
reaction volume, in lanes 05, respectively). Lane MW, 100 bp molecular
weight marker (MBI Fermentas).

10 bone samples while the fragment was absent in both the

extraction (E) and amplification (B) controls (Fig. 2B). In the
isopropanol precipitation method due to the presence of
remaining inhibitors two consecutive PCR amplifications with
40 cycles each were needed to obtain sufficient amounts of
PCR product for sequencing, while in our technique only one
round of PCR amplification was needed with 35 cycles. This
considerably reduced the chance of contamination and
provided the successful removal of the inhibitors.
The unsuccessful amplification of the partial overlapping
406 bp mtDNA fragment from the hypervariable region I (data
not shown) also supports the statement that the 239 bp
fragment genuinely came from the ancient DNA. The amplified
239 bp PCR products were successfully sequenced in both
iii

e67

Nucleic Acids Research, 2000, Vol. 28, No. 12

REFERENCES

Figure 2. PCR amplification of the human mitochondrial hypervariable

region I (positions 1618216420) from ancient bone samples with 35 cycles.
(A) Amplification of a 239 bp fragment from ancient bone no. 3 after the
isopropanol-based or the Dextran Blue mediated extraction methods. Lane A,
PCR positive control from a blood sample; lanes 05, 05 l bone as
template; lane MW, molecular weight marker (pBR322/HaeIII); lane M, PCR
amplification mix control. (B) Test for the amplification authenticity of the
ancient DNA extracts. Lane A, PCR positive control from a blood sample;
lanes 113, ancient bone samples no. 11no. 3; lane MW, molecular weight
marker (pBR322/HaeIII); lane B, amplification control; lane M, PCR
amplification mix control; lanes E1 and E2, the extraction controls.

directions in all cases and showed mitochondrial identity

(GenBank accession numbers AF228540AF228549).
The simple and efficient carrier-mediated ethanol precipitation
protocol presented here could be useful for PCR amplifiable
DNA extraction from ancient remains of excavated burial sites
where the previously published methods failed.
ACKNOWLEDGEMENTS
The authors would like to thank Mrs Mria Rad and Mrs
Gabriella Lehcz for skilled technical assistance; Dr S. J. Devlin
for critical reading of the manuscript; for the support and
encouragement of Lajos Keszthelyi during the entire project.
This work was supported by grants OTKA T021318 from the
Hungarian National Scientific Research Fund to I.R.
iv

1. Hagelberg,E., Sykes,B. and Hedges,R. (1989) Nature, 342, 485.

2. Hnni,C., Brousseau,T., Laudet,V. and Stehelin,D. (1995) Nucleic Acids Res.,
23, 881882.
3. Tuross,N. (1994) Experientia, 50, 530535.
4. Pbo,S. (1990) PCR Protocols: A Guide to Methods and Applications.
Academic Press, Inc., San Diego, CA, pp. 159166.
5. Scholz,M. and Pusch,C.M. (1997) Technical Tips Online, T01045.
6. Hagelberg,E., Bell,L.S., Allen,T., Boyde,A., Jones,S.J. and Clegg,J.B.
(1991) Philos. Trans. R. Soc. Lond. B. Biol. Sci., 333, 399407.
7. Hnni,C., Begue,A., Laudet,V. and Stehelin,D. (1995) J. Archaeological Sci.,
22, 649658.
8. Pbo,S., Higuchi,R.G. and Wilson,A.C. (1989) J. Biol. Chem., 264,
97099712.
9. Pbo,S. (1989) Proc. Natl Acad. Sci. USA, 86, 19391943.
10. Hoss,M., Jaruga,P., Zastawny,T.H., Dizdaroglu,M. and Pbo,S. (1996)
Nucleic Acids Res., 24, 13041307.
11. Scholz,M., Giddings,I. and Pusch,C.M. (1998) Anal. Biochem., 259, 283286.
12. Lindahl,T. (1993) Nature, 362, 709715.
13. Meijer,H., Perizonius,W.R. and Geraedts,J.P. (1992) Biochem. Biophys.
Res. Commun., 183, 367374.
14. Fisher,D.L., Holland,M.M., Mitchell,L., Sledzik,P.S., Wilcox,A.W.,
Wadhams,M. and Weedn,V.W. (1993) J. Forensic Sci., 38, 6068.
15. Lee,H.C., Pagliaro,E.M., Berka,K.M., Folk,N.L., Anderson,D.T.,
Ruano,G., Keith,T.P., Phipps,P., Herrin,G.L.,Jr and Garner,D.D. (1991)
J. Forensic Sci., 36, 320330.
16. Cattaneo,C., Smillie,D.M., Gelsthorpe,K., Piccinini,A., Gelsthrope,A.R.
and Sokol,R.J. (1995) J. Forensic Sci., 74, 167174.
17. Kurosaki,K., Matsushita,T. and Ueda,S. (1993) Am. J. Hum. Genet., 53,
638643.
18. Faerman,M., Filon,D., Kahila,G., Greenblatt,C.L., Smith,P. and
Oppenheim,A. (1995) Gene, 167, 327332.
19. Perry,W.L.3., Bass,W.M., Riggsby,W.S. and Sirotkin,K. (1988)
J. Forensic Sci., 33, 144153.
20. Blake,E., Mihalovich,J., Higuchi,R., Walsh,P.S. and Erlich,H. (1992)
J. Forensic Sci., 37, 700726.
21. Cattaneo,C., Craig,O.E., James,N.T. and Sokol,R.J. (1997) J. Forensic
Sci., 42, 11261135.
22. Yang,D.Y., Eng,B., Waye,J.S., Dudar,J.C. and Saunders,S.R. (1998)
Am. J. Phys. Anthropol., 105, 539543.
23. Hoss,M. and Pbo,S. (1993) Nucleic Acids Res., 21, 39133914.
24. Evison,M.P., Smillie,D.M. and Chamberlain,A.T. (1997) J. Forensic Sci.,
42, 10321038.
25. Prado,V.F., Castro,A.K., Oliveira,C.L., Souza,K.T. and Pena,S.D. (1997)
Genet. Anal., 14, 4144.
26. Walsh,P.S., Metzger,D.A. and Higuchi,R. (1991) Biotechniques, 10, 506513.
27. Schnee-Griese,J. and Linder,S. (1994) In Bar,W., Fiori,A. and Rossi,U.
(eds), Advances in Forensic Haemogenetics. Springer Verlag, Berlin,
Germany, pp. 167169.
28. Pusch,C.M., Giddings,I. and Scholz,M. (1998) Nucleic Acids Res., 26,
857859.
29. Pusch,C.M. and Scholz,M. (1997) Technical Tips Online, T01217.
30. H.Tth,E. (1994) M5 a tutplya 72. lelhely avar temetjnek feltrsa
Kecskemt. Mzeumi Kutatsok Bcs-Kiskun Megyben, Bcs-Kiskun
Megyei nkormnyzat Mzeumi Szervezete, Kecskemt, Hungary,
pp. 153160.
31. Bdi,G. (1996) Thesis: A hetnyegyhzi avarkori szria embertani
feldolgozsa. Department of Anthropology, University of Attila Jzsef,
Szeged, Hungary.
32. Olh,S. (1990) Thesis: Srrtudvar-Hzfld honfoglalskori temetjnek
embertani rtkelse. Department of Anthropology, University of Attila
Jzsef, Szeged, Hungary.
33. B.Nagy,K. (1993) In Lorinczy Gyula (ed.), Az Alfld 9. szzadban.
Szeged, Hungary, pp. 151171.
34. Matysiak-Scholze,U., Dimmeler,S. and Nehls,M. (1996) Technical Tips
Online, T40011.
35. Anderson,S., Bankier,A.T., Barrell,B.G., de Bruijn,M.H., Coulson,A.R.,
Drouin,J., Eperon,I.C., Nierlich,D.P., Roe,B.A., Sanger,F. et al. (1981)
Nature, 290, 457465.
36. Wilson,M.R., Polanskey,D., Butler,J., DiZinno,J.A., Replogle,J. and
Budowle,B. (1995) Biotechniques, 18, 662669.